Synthesis and degradation of proteins in cultured,androgen-responsive tumour cells

Changes in the rates of de novo synthesis of proteins and in the rates of degradation of proteins were studied in cultured androgen-responsive tumour cells. The proliferation rate of these cells is regulated both by cell population density and by physiological concentrations of androgens, such as testosterone. Both rates of de novo synthesis and rates of degradation of proteins changed with proliferation rate, although neither were directly proportional to proliferation rate. By contrast, the net rate of protein accumulation was always directly proportional to proliferation rate. This relationship held despite the fact that the mean amounts of protein and RNA per cell changed with density. These results suggest that, under certain conditions, a change in the net rate of protein accumulation may be sufficient to change proliferation rate.     

Effect of diaminobutene and diaminopropane on diet-stimulated polyamine synthesis and cell proliferation in rat liver.

The effect of two putrescine analogs were studied on hepatic polyamine synthesis and cell proliferation, both of which were stimulated by food intake. Trans-1, 4-diamino-2-butene (diaminobutene), which is a potent competitive inhibitor of ornithine decarboxylase [EC 4.1.1.17] (ODC), repressed the induction of ODC and effectively inhibited the accumulation of putrescine in rat liver which was induced by the feeding of dietary protein. Unexpectedly, diaminobutene did not suppress DNA synthesis and mitotic activity in rat liver, suggesting that it can mimic the role of putrescine in cell proliferation. 1,3-Diaminopropane effectively repressed the induction of ODC caused by food intake and also suppressed DNA synthesis and mitotic activity without affecting the accumulation of RNA or protein. The suppression of mitotic activity by 1,3-diaminopropane was reversed by a single injection of putrescine, spermidine, spermine, or diaminobutene. It was concluded that rapid accumulation of polyamines, especially putrescine, was a prerequisite for the later enhancement of DNA synthesis and cell proliferation in rat liver caused by food intake.     

The relationship of DNA synthesis to protein accumulation in the cell nucleus

The relationship between DNA synthesis and protein accumulation in cell nucleus and cytoplasm has been investigated by the use of a combination of ultramicrointerferometric and ultramicrospectrophotometric methods. 5-Fluoro-2'-deoxyuridine (FUdR) inhibited DNA synthesis, resulting in inhibition of cell proliferation in G-1 and early S-phase. However, synthesis and accumulation of protein continued in the presence of FUdR, as indicated by a 54% increase in the average dry mass value per individual cell during 18-hour exposure to FUdR; due primarily to protein accumulation in the cytoplasm, the average cytoplasmic dry mass increased by as much as 85%, while the dry mass of the nucleus increased by only 21%. The dry mass values of individual nuclei were well-correlated to the nuclear DNA content throughout the period of exposure to FUdR. In contrast to the continued accumulation of protein in the cytoplasm during inhibition of DNA synthesis, protein accumulation in the nucleus was inhibited. When cells were released from inhibition of DNA synthesis by the addition of 2'-deoxythymidine, the nuclear DNA content and nuclear dry mass increased in near-synchrony, there being some evidence that DNA synthesis was initiated somewhat prior to initiation of increase in nuclear dry mass. Thus, it appears that DNA synthesis (or an increase in nuclear DNA content) is intimately related to the regulation of protein accumulation in the nucleus.     

Targeting the retinoblastoma protein by MC007L, gene product of the molluscum contagiosum virus: detection of a novel virus-cell interaction by a member of the poxviruses

The human pathogenic poxvirus molluscum contagiosum virus (MCV) is the causative agent of benign neoplasm, with worldwide incidence, characterized by intraepidermal hyperplasia and hypertrophy of cells. Here, we present evidence that the MC007L protein of MCV targets retinoblastoma protein (pRb) via a conserved LxCxE motif, which is present in many viral oncoproteins. The deregulation of the pRb pathway plays a central role in tumor pathogenesis. The oncoproteins of small DNA viruses contain amino acid sequences that bind to and inactivate pRb. Isolated expression of these oncoproteins induces apoptosis, cell proliferation, and cellular transformation. The MC007L gene displays no homology to other genes within the poxvirus family. The protein anchors into the outer mitochondrial membrane via an N-terminal mitochondrial targeting sequence. Through the LxCxE motifs, MC007L induces a cytosolic sequestration of pRb at mitochondrial membranes, leading to the inactivation of the protein by mislocalization. MC007L precipitates the endogenous pRb/E2F-1 complex. Moreover, MC007L is able to cooperate to transform primary rat kidney cells. The interaction between MC007L and pRb provides a novel mechanism by which a virus can perturb the cell cycle.     

Myc and Max function as a nucleoprotein complex.

The Myc family of oncoproteins are thought to regulate proliferation and differentiation in a wide variety of cell types. Recent studies show that Myc proteins form sequence-specific DNA-binding complexes with Max, a new member of the helix-loop-helix leucine zipper protein class. The properties of the Myc-Max complex suggest a mechanism for Myc's function in both normal and neoplastic cell behavior.     

Hypertrophy of cultured bovine aortic endothelium following irradiation

The vascular endothelium is a vital multifunctional tissue which covers the entire luminal surface of the circulatory system. Loss of continuity of the endothelial lining normally results in cell migration and proliferation to make up for cell loss and to ensure that exposure of the thrombogenic subendothelium to platelets and clotting factors is minimized. We showed that ionizing radiation (400-3000 cGy) causes dose-dependent cell loss from confluent monolayer cultures of bovine aortic endothelium, which cannot immediately be compensated by cell proliferation. Within 24 h, the remaining attached cells undergo substantial somatic hypertrophy (evidenced by increased protein content, cell volume, and attachment area) but remain diploid. If cell loss is not excessive, monolayer continuity is restored within several days. Although reduced protein degradation may contribute, most of the protein accumulation is due to synthesis of new protein. Unlike endothelium, irradiation of smooth muscle cultures causes neither cell loss nor increased protein synthesis. Hypertrophy of irradiated endothelial cells appears to be a consequence of a proliferative stimulus (cell loss) in a population of cells which is unable to divide. It can be modulated by replating irradiated cells at different densities. We suggest that endothelial hypertrophy is an early vascular homeostatic response before clonal proliferation of surviving cells or repopulation by cells from outside of the irradiated field can compensate for cell loss.     

Mechanism of direct cell interactions. Self-organization of protein synthesis rhythm

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 μM or forskolin at 10 μM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 μM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 μM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists → changes in the level of calcium ions in cytoplasm → increased protein kinase activities → protein phosphorylation → modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.     

Competitive binding of AUF1 and TIAR to MYC mRNA controls its translation

(A+U)-rich elements (AREs) within 3' untranslated regions are signals for rapid degradation of messenger RNAs encoding many oncoproteins and cytokines. The ARE-binding protein AUF1 contributes to their degradation. We identified MYC proto-oncogene mRNA as a cellular AUF1 target. Levels of MYC translation and cell proliferation were proportional to AUF1 abundance but inversely proportional to the abundance of the ARE-binding protein TIAR, a MYC translational suppressor. Both AUF1 and TIAR affected MYC translation via the ARE without affecting mRNA abundance. Altering association of one ARE-binding protein with MYC mRNA in vivo reciprocally affected mRNA association with the other protein. Finally, genetic experiments revealed that AUF1 and TIAR control proliferation by a MYC-dependent pathway. Together, these observations suggest a novel regulatory mechanism where tuning the ratios of AUF1 and TIAR bound to MYC mRNA permits dynamic control of MYC translation and cell proliferation.     

Cell context in the control of self-renewal and proliferation regulated by MLL1

Mixed lineage leukemia 1 (MLL1) is a gene that is disrupted by chromosomal translocation characteristically in a large proportion of infant leukemia and also in a fraction of childhood and adult leukemia. MLL1 encodes a chromatin regulatory protein related to the Drosophila Trithorax protein, a well-studied epigenetic factor that functions during development to maintain expression of its target genes. Although tremendous progress has been made understanding the downstream targets of MLL1 fusion oncoproteins and how manipulation of those targets impacts leukemogenesis, very little is known regarding how the initial expression of an MLL1 fusion protein impacts on that cell’s behavior, particularly how the cell cycle is affected. Here, we focused on the function of endogenous MLL1 in the stem and progenitor cell types that are likely to be transformed upon MLL1 translocation. Our studies reveal a differential response of stem or progenitor populations to acute loss of MLL1 on proliferation and survival. These data suggest that the effects of MLL1 fusion oncoproteins will initiate the leukemogenic process differentially depending on the differentiation state of the cell type in which the translocation occurs.     

Role of endogenous TNF-alpha and sphingosine in induced DNA synthesis in regenerating rat liver after partial hepatectomy.

Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover.     

泛素连接酶SCF~(FBW7)调控的靶蛋白研究进展

FBW7(F-box and WD repeat domain-containing7,FBW7)为F-box蛋白家族成员,是SCF型泛素连接酶复合物的底物识别蛋白,介导细胞内多种蛋白质经泛素-蛋白酶体途径降解。FBW7通过靶向降解多种癌蛋白如Mcl-1、Notch、HIF-1α、Cyclin E和KFL5等,参与调控细胞增殖、分化、凋亡及肿瘤转移等多种生物学过程。作为一种肿瘤抑制蛋白,FBW7基因突变或缺失存在于多种人类肿瘤中,如白血病、乳腺癌、卵巢癌、胆管癌等,并在这些肿瘤的发生和发展中发挥重要作用。因此,针对FBW7的深入研究有助于理解肿瘤的发生发展机制以及开发肿瘤治疗新方案。本文就FBW7调控的癌蛋白研究进展作一综述。     

RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells

The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.     

Opposite effects of glucocorticoid on estrogen-induced growth and differentiation of quail oviduct: demonstration by sequential treatments

Control of the development and functions of avian oviduct is monitored by four classes of steroid hormones, including glucocorticoids. The effects of dexamethasone (DEX), a synthetic glucocorticoid, were studied via sequential treatments with estradiol benzoate, paying special attention to changes in estrogenic oviduct responses involving DNA synthesis and cell proliferation, ovalbumin accumulation and cell differentiation. DEX exerted an antagonistic effect upon estrogen stimulation when administered separately before or after estradiol benzoate (EB). Given before EB, DEX was more strongly antagonistic for DNA synthesis than when given simultaneously with EB. Administered after EB, DEX reversed EB-induced cell proliferation: the DNA content declined and the oviduct regressed. In the same way, protein and ovalbumin synthesis was inhibited and delayed by first intervention of DEX, and accelerated catabolism of ovalbumin and proteins was observed when DEX followed EB. DEX, which was ineffective alone, but synergistic on ovalbumin synthesis when given concomitantly with EB, prevented or dissipated the estrogenic effects, cell proliferation and secretory process when administered in sequential treatments.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

Down-regulation of AP1 activities after polarization of vas deferens epithelial cells correlates with androgen-induced gene expression

Vas deferens epithelial cell subcultures were used to study the sequential regulation of jun/fos proto-oncogene expression and AP1 activities during cell proliferation, polarization and androgen-induced expression of a terminal differentiation marker, i. e. the mvdp gene. Proliferation of epithelial cells is associated with a high expression in the nucleus of most Jun and Fos oncoproteins. After cell seeding on an extracellular matrix which allows polarization and expression of the mvdp gene in response to androgens, AP1 protein accumulation is greatly altered and consists in a loss of JunB, Fra1, FosB and a decrease in c-Fos, c-Jun and Fra2, while JunD remained at the same level. This was correlated with a drop in AP1 binding activity as evaluated by gel shift assay using either AP1 consensus sequence or AP1 binding sites of the mvdp gene promoter region, and in AP1 transactivating activity, as estimated by stable transfection experiments using an AP1 responsive promoter (TRE-TK-luc). Androgens did not significantly influence AP1 activities. On the contrary, stimulation of AP1 proteins by the tumor-promoting phorbol ester caused a decrease in androgen-induced mvdp mRNA accumulation, and this effect was reversed by staurosporine, a potent inhibitor of PKC. Our data suggest that a down-regulation of AP1 activities induced by epithelial cell differentiation is a prerequisite to androgen-induced mvdp gene expression. The high AP1 activities observed during proliferative state or induced in TPA-treated polarized cells, exert a repressive effect on androgen action.